This invention provides a simple and economical method for producing hybrid seeds by controlling micronutrient content of the male reproductive organs. More specifically, the invention relates to a method for inducing cross-pollination in plant species such as wheat that are normally self-pollinating, by selecting parents having differential fertility when grown in micronutrient-deficient growth media, and crossing them.
Hybrid seeds, those which are genetically heterozygous, have many advantages over homozygous seeds. Hybrid plants commonly grow faster, achieve higher biomass and yield, and have greater disease resistance than their better parent. This phenomenon is known as heterosis.
Plant hybridization is a crowded and mature art, but there has long been a need for effective, simple, economical methods for breeding hybrid wheat and other small-grained self-fertilizing plants. Hybrids are made by inter-crossing separate inbred lines. Generally, a breeder introduces viable donor pollen from a male fertile line onto the potentially fertile stigmas of a male sterile line that lacks fertile pollen. Genetic, mechanical, chemical, and biotechnological processes have been used to induce male sterility to facilitate hybrid seed production.
Genic male sterility has been found in barley. (Suneson, C. A., "A male sterile character in barley," J. Heredity. 31:213-214 (1940)), and wheat (Suneson, C. A., "Use of Pugsley s sterile wheat in cross breeding," Crop Sci. 2:534-535 (1962)). However, since plants with the male sterile genotype are self-infertile, they (and hence the male sterile genes) can only be maintained in heterozygous populations. E.g. see Briggle, L. W., "A recessive gene for male sterility in hexaploid wheat," Crop Sci. 10:693-696 (1970); Gill, B. S. and Anand, S. C., "Genetic male sterility for hybrid seed production in wheat," Crop Sci. 10:385-386 (1970). This has made it necessary to develop complicated breeding procedures to make use of genic male sterility. E.g. Suneson, C. A., "The use of male-sterility in barley improvement," J. Am. Soc. Agron. 37:72-73 (1945); Driscoll, C. J., "X Y Z system of producing hybrid wheat," Crop Sci. 12:516-517 (1972); and "Modified X Y Z system of producing hybrid wheat," Crop Sci. 25:1115-1116 (1985).
Cytoplasmic-genetic male sterility and fertility restoration requires breeding a male sterile line which retains female-fertile characteristics. Allard, R. W. Principles of Plant Breeding (John Wiley & Sons 1960), pp. 243-251. A plant that is considered to have desirable characteristics and is to be the female parent needs to go through extensive inbreeding through many generations over as many as ten years to be made male sterile. In the process of producing hybrids two other breeding programs have to be running concurrently. One is required to restore the fertility of the male sterile line when the hybrid is growing, so that it can be raised for further hybridization. The other is to introduce the male sterile gene or male sterile cytoplasm. The sources of male-sterility and the restorers are often unreliable. The method of using cytoplasmic genetic male sterility and fertility restoration systems was developed to produce hybrid rice seeds in bulk in China. (Yuan and Virmani, 1988). It has also been used for alfalfa. U.S. Pat. No. 3,570,181. Another type of genetic pollination control is nuclear genic male sterility. This has been proposed to develop hybrid wheat parents. Lucken, K. A. and Johnson, K. D., "Hybrid wheat status and outlook" in Hybrid Rice (International Rice Research Institute 1988), pp. 243-255. In another approach, environment-sensitive genic male sterile rice lines are sterile under certain daylength and temperature conditions, but fertile under other conditions. Lu et al., "Current status of two-line method of hybrid rice breeding," Hybrid Rice Technology (IRRI 1994), pp. 37-49. These may be used in a two-line breeding method, but it is difficult to control growing conditions suitably to provide the desired degree of fertility or sterility.
The mechanical approach involves manual emasculation of floral structures. The male part is removed manually, and then the female part of the flower is cross-fertilized with pollen (male gametes) from another plant. This approach is feasible in plants with large flowers with male and female parts located separately such as corn, but many important agricultural plants such as wheat and rice have very small flowers in which the male and female parts are located very close together. The mechanical approach is labor intensive, time-consuming, and inefficient, limiting the number of flowers that can be manually cross-fertilized and the number of crosses made. It has been impossible to explore the characteristics and advantages of many gene combinations due to these limitations.
Chemical approaches have been employed in which a gametocidal composition is applied to the anthers of a plant to induce sterility of the male organ. The gametocidal composition may comprise cinnoline compounds (EP 363236, U.S. Pat. No. 5,129,939), azetidine derivatives (EP 29265, U.S. Pat. No. 4,555,260), polychloroacetic acids and their derivatives (SU 641926), amega-amino-carboxylic acids (SU 635929), tetra-chloroalkane derivatives (SU 635928), and the like. However, chemical approaches are often costly and may produce undesirable side effects, for example retardation of plant growth, and poor seed set. Furthermore, chemical hybridizing agents have significant negative environmental impacts and so some countries have banned them.
Recombinant DNA technology has also been developed to produce male sterile plants. For example Albertsen et al. (AU 9337990) disclose a method of providing heritable, externally-controllable male-sterility in a plant, by inactivating a flavonol-producing gene. In another application (EP 513884), a method of inducing male-sterility by inhibiting the expression of a gene encoding an enzyme in chalcone biosynthesis is reported. There are several problems associated with these approaches, including the restriction of such technology to readily-transformable crop species, the time taken to obtain transgenic crops and the small scale of operations relating to recombinant DNA technologies. Furthermore, the present state of the art can only address one or two specific problems at a time. Approaches utilizing traditional plant breeding have the same limitations and require many generations of back-crossing to remove undesirable traits.
A significant disadvantage of these prior approaches to producing hybrid seeds is their high cost. Thus, efforts to hybridize wheat and other small grained self-fertilizing plants simply, effectively, and economically have failed, and there is a need for an economical method for mass-producing hybrid wheat and rice and similar plants.
In a different field of study, it was reported long ago that copper deficiencies can cause male sterility in wheat but this observation did not lead to any viable method for hybridizing wheat. Graham, R. D., "Male sterility in wheat plants deficient in copper," Nature 254:514-515 (1975). Deficiencies of other micronutrients have been found to cause male sterility in other plant species, including manganese, zinc and molybdenum deficiency in corn. C. P. Sharma, P. N. Sharma, C. Chatterjee, and S. C. Agarwalla, "Manganese deficiency in maize affects pollen viability," Plant and Soil, 138:139-142 (1991); P. N. Sharma, C. Chatterjee, S. C. Agarwalla, and C. P. Sharma, "Zinc Deficiency and pollen fertility in maize (Zae mays)", Plant and Soil, 124:221-225 (1990); S. C. Agarwalla, C. Chatterjee, P. N. Sharma, C. P. Sharma, and N. Nautiyal, "Pollen development in maize plants subjected to molybdenum deficiency," Can. J. Bot., 57:1946-1950.
Boron-deficient soils occur naturally throughout the world. Soil boron may be depleted by repeated cropping. Liming, a routine soil amendment in agriculture, can also decrease the amount of boron that is available to plants. Research in this area has been directed toward understanding the interaction of boron deficiency and environmental factors such as temperature, humidity, and light. Efforts have also been made to diagnose the mechanisms of sterility, to find crops that can grow well and develop viable pollen in boron-deficient soil, and to restore fertility by applying boron. Rerkasem, B., Netsangtip, R., Lordkaew, S., Cheng, C., "Grain set failure in boron deficient wheat," Plant and Soil 155/156:309-312 (1993); Cheng, C. and Rerkasem, B., "Effects of boron on pollen viability in wheat," Plant and Soil 155/156:313-315 (1993).
Genotypic differences in the response to micronutrient deficiency have also been reported in different species. This research is directed toward finding plants that are growth and yield tolerant of the deficiency, or to understanding the underlying biochemistry of fertility. For example, for copper deficiency in wheat, rye and triticale, see E. K. S. Nambiar, "Genetic differences in the copper nutrition of cereals. I. Differential response of genotypes to copper," Aust. J. Agric. Res., 27:453-463 (1976); R. D. Graham, and D. T. Pearce, "The sensitivity of hexaploid and octaploid triticales and their parent species to copper deficiency", Aust. J. Agric. Res., 30:791-799 (1979); and Marschner (1992). For manganese deficiency in barley, see W. Ralph, "Managing manganese deficiency," Rural Research, 130:18-22 (1986); and N. E. Marcar, and R. D. Graham, "Genotypic variation for manganese deficiency in wheat," J. Plant Nutrition, 10:2049-2055 (1987). For zinc deficiency in wheat, see R. D. Graham, J. S. Ascher and S. C. Hynes, "Selecting zinc-efficient cereal genotypes for soils of low zinc status," Plant and Soil, 146:241-250 (1992). For zinc deficiency in soybean, see E. E. Hartwig, W. F. Jones, T. C. Kilen, "Identification and inheritance of inefficient zinc absorption in soybean," Crop Sci., 31:61-63 (1991).
Likewise, the effects of boron deficiency on fertility in wheat and barley vary among genotypes. Rerkasem, B. and Jamjod, S., "Correcting boron deficiency induced ear sterility in wheat and barley," Thai Jour. Soils and Fertilizers 11:200-209 (1989). For example, when raised in medium having a low boron level, wheat line SW41 was self-infertile when bagged. Also, it was found that a wheat line raised in boron deficient medium had marginal fertility even when manually cross-fertilized by pollen from a fertile male. Rerkasem et al., Plant and Soil 155/156:309-312 (1993).
Male plant sterility induced by micronutrient deficiency has been viewed as a major disadvantage and an undesirable trait reducing the productivity of crops. There has been no suggestion of how to use this undesirable phenomenon in a productive fashion. In particular, past research does not suggest using micronutrient deficiency to provide a fertility-selective growth medium, and a method of selecting a female line which is micronutrient deficiency tolerant as to female fertility and micronutrient deficiency sensitive as to male fertility, and a male line that is micronutrient deficiency tolerant as to male fertility, and allowing cross-fertilization to occur between them. Most specifically, past research has not suggested a simple method to produce hybrids of small grains at field scale, thus lowering the cost of production and making hybrid seeds cost-effective for small grains.
The mechanisms for fertility differences are not known. The degree of male sterility in a plant is not indicated by boron concentrations in the soil, or in leaves and whole flowers (Rerkasem B. and S. Lordkaew, "Predicting grain set failure with tissue boron analysis," in Mann C. E and B. Rerkasem (eds.), Boron Deficiency in Wheat. pp. 9-14, CIMMYT Wheat Special Report No. 11. CIMMYT, Mexico, 1992; Rerkasem and Loneragan, 1994). At the general level, boron uptake, movement and transportation into a plant can be controlled by methods known to those familiar with the science of plant nutrition (Marschner, H., "Mineral Nutrition of Higher Plants," Academic Press, transportation into a plant can be controlled by methods known to those familiar with the science of plant nutrition (Marschner, H., "Mineral Nutrition of Higher Plants," Academic Press, London, 1995; Mortvedt, J. J., F. R. Cox, L. M. Shuman, R. M. Welch, eds. "Micronutrients in Agriculture," Soil Sci. Soc. Amer. Book Series No. 4, SSSA, Madison, Wis., 1991). However, the possibility of using boron levels for precise control of male fertility/sterility was not previously recognized and was not accomplished prior to this invention.